Read-through compounds have received much attention recently. Certain compounds such as gentamicin and PTC124 have been demonstrated with ability to induce readthrough of PTCs, which allows translation of full-length protein. In many cases, the readthrough induced full-length protein is at least partially functional, even when it contains a mis-incorporated amino acid. Therefore, chemical-induced readthrough of PTCs might be exploited as a potential treatment strategy for genetic disorders caused by nonsense mutations.
Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)-enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model (Du et al, 2009). This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS. The assay is luciferase independent. We screened about 34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a natural nonsense mutation and induced significant amounts of dystrophin protein.
Translation termination is signaled by three stop codons: UAA, UAG, and UGA. This mechanism is highly conserved, although each stop codon has a different efficiency for terminating translation. UGA is considered to be a “leaky” stop codon with the highest intrinsic readthrough potential. UAA shows high fidelity and little intrinsic readthrough potential, whereas UAG has intermediate fidelity (see, e.g., Weiner and Weber, 1973, J. Mol. Biol. 80:837-855). Nonsense mutations create primary premature termination codons (PTCs) and result in either no formation of the target protein or truncated protein with impaired stability.
Certain compounds influence the fidelity of stop codon recognition and induce readthrough of primary PTCs, which allows translation of some full-length protein. In many cases, the readthrough-induced protein is functional, even when it contains a wrongly incorporated amino acid (Keeling and Bedwell, 2005, Current Pharmacogenomics. 3:259-269; Zingman et al., 2007, Clin. Pharmacol. Ther. 81:99-103).
It is estimated that 30% of human disease-causing alleles are nonsense mutations (Mendell and Dietz, 2001; Du et al., 2009, JEM, 206 (10): 2285). Other types of mutation, such as frameshift and splicing mutations, lead to secondary PTCs; however, these are not therapeutic targets for readthrough compounds (RTCs). Considering that >1,800 distinct genetic disorders are caused by nonsense mutations, the readthrough of primary PTCs has treatment potential for large numbers of patients.
To date, there is no efficient treatment for a majority of genetic disorders, such as ataxia telangiectasia (A-T) and Duchenne Muscular Dystrophy (DMD). It has been demonstrated that certain compounds could induce readthrough of premature termination codons (PTCs), which allows translation of full-length protein. In many cases, the readthrough-induced protein is at least partially functional, even when it contains an inaccurate incorporated amino acid (Lai et al., 2004; Du et al., 2009; Keeling and Bedwell, 2005; Zingman et al., 2007). Therefore, chemical-induced readthrough of PTCs might be exploited as a potential treatment strategy. Notably, this readthrough strategy could be especially useful for diseases like A-T and cystic fibrosis (CF), because a small amount of protein restored by readthrough compounds (RTCs), even as little as 1-10%, may still be able to significantly reduce the severity or eliminate the principal manifestations of these disease (Gilad S, 1998; Kerem, 2004; Ramalho et al., 2002, Chun et al., 2004; Du et al., 2009; Kayali et al., 2012).
To date, most reported PTC-RTCs that are active in mammalian cells have belonged to the aminoglycoside antibiotics class (e.g., gentamicin, paromomycin, G418 and its derivatives NB74 and NB84) (Keeling and Bedwell, 2005; Zingman et al., 2007). Certain types of aminoglycosides can induce ribosomes to read through PTC mutations via insertion of a random amino acid by near-cognate transfer RNA. The therapeutic potential of aminoglycosides has been evaluated in the laboratory for different genetic models, such as cystic fibrosis (see, e.g., Du et al., 2002, J. Mol. Med. 80:595-604; Howard et al., 1996; Bedwell et al., 1997), muscular dystrophy (see, e.g., Loufrani et al., 2004, Arterioscler. Thromb. Vasc. Biol. 24:671-676; Howard et al., 2000; Loufrani et al., 2004), Hurler syndrome (Keeling et al., 2001, Hum. Mol. Genet. 10:291-299), cystinosis (Helip-Wooley et al., 2002, Mol. Genet. Metab. 75:128-133), spinal muscular atrophy (Sossi et al., 2001, Eur. J. Hum. Genet. 9:113-120), ataxia-telangiectasia (Lai et al., 2004, Proc. Natl. Acad. Sci. USA. 101:15676-15681), and type 1 Usher syndrome (Rebibo-Sabbah et al., 2007, Hum. Genet. 122:373-381) and such models could be used to evaluate the compounds described herein. Clinical trials also indicate that aminoglycosides can induce some functional protein production; however, the therapeutic benefits remain uncertain (see, e.g., Politano et al., 2003, Acta Myol. 22:15-21; Wilschanski et al., 2000; Clancy et al., 2001). Furthermore, the systemic toxicity of most commercial aminoglycosides in mammals has greatly diminished their potential for successful readthrough therapy (Mingeot-Leclercq and Tulkens, 1999, Antimicrob. Agents Chemother. 43:1003-1012; Guan et al., 2000, Hum. Mol. Genet. 9:1787-1793). Therefore, efforts are underway to develop better aminoglycoside derivatives with reduced toxicity and enhanced activity (Nudelman et al., 2006, Bioorg. Med. Chem. Lett. 16:6310-6315; Rebibo-Sabbah et al., 2007, Hum. Genet. 122:373-381).
Nonaminoglycosides (e.g. PTC124, RTC13, RTC14 and tylosin) have been described as well. The macrolide Tylosin has RT activitiy in prokaryotes and is being further evaluated in patients with somatic mutations in colon cancer (Zilberberg et al 2010). Recently, PTC Therapeutics (South Plainfield, N.J.) described a nonaminoglycoside RTC, PTC124, which was developed synthetically by screening >800,000 chemicals and analogues using a luciferase-based HTS assay (see, e.g., Welch et al., 2007, Nature. 447:87-91; Du M et al., 2008). A phase-I clinical study in cystic fibrosis showed that PTC124 is generally well tolerated and appears to have more efficient readthrough activity than aminoglycosides (Hirawat et al., 2007, J. Clin. Pharmacol. 47:430-444). Moreover, PTC124 does not induce ribosomal readthrough of normal stop codons. However, PTC124 is not equally effective with all three stop codons, working best on the TGA stop codon (Welch et al., 2007). This selective activity limits the number of patients who could be treated with PTC124. Further, PTC124's recent phase 2b clinical study in DMD patients failed with participants not showing a significant improvement in the six-minute walk distance or dystrophin expression (Guglieri M and Bushby K, 2010). In addition, a recent study indicates that the initial discovery of PTC124 by HTS may have been biased due to its direct interaction with the FLuc (firefly luciferase) reporter used (Auld et al., 2009, Proc. Natl. Acad. Sci. USA. 106:3585-3590 Peltz et al. 2009; Auld et al. 2010), indicating the importance of a luciferase-independent HTS assay for future drug screening. Lastly, PTC124 does not effectively cross the blood-brain barrier, a critical factor for treating neurological disorders like A-T, Alzheimer diseases and the CNS effects encountered in Hurler patients.
Therefore, successfully developing new RTCs with optimized efficacy and low toxicity could benefit numerous genetic diseases which are caused by nonsense mutations and currently have no cure. Notably, the potential therapeutic benefit of a small molecular RTC may also extend to cancer for individuals carrying nonsense mutations in genes such as BRAC1, BRAC2 and CHEK2. The RTCs described herein are useful for targeting nonsense mutations that cause numerous disorders and diseases. Therefore, the discovery of these new RTCs is a very important finding in this field.